We have a strange result: We transduce neurons with an AAV vector with synapsin promoter then DIO-ChrimsonR-mRuby - meaning there are double loxP-lox2722 sites flanking the inverted ChrimsonR-mRuby fusion construct. We then introduce a plasmid by single-cell electroporation in a lentiviral backbone that has cre-recombinase and everything was great - huge photocurrents from the opsin in the red fluorescent neurons. Now we package the second plasmid into an Ecolenti (i.e. a lentiviral vector with a serotype that only enters mouse cells) and we have red fluorescent neurons with no photocurrents. My suspicion is that the ChrimsonR must be mutated or something - the promoter and a start codon must be active or there would be no red fluorescence but it is very strange that the first part of the protein is now non-functional. I want to PCR and sequence the DNA but I am very challenged when it comes to designing primers that will be specific for the DNA in cells with active cre as I don't want to also amplify from the cells in which the non-recombined plasmid is. Anyone know a forward primer that would be specific for the inverted and excised DNA? Many thanks! Chris